Mycobacterium tuberculosis (Mtb), the main etiologic agent of human tuberculosis (TB), remains a leading cause of worldwide morbidity and mortality. The percentage of HIV positive diagnosis among new TB cases is on the rise and the risk of developing TB is at least 20 times higher in HIV positive compared to HIV negative individuals. The emergence of Mtb strains highly resistant to chemotherapy is also a major concern to AIDS patients. In this context, our long term goal is to perform molecular genetics and biochemical analyses of the D-alanine pathway of peptidoglycan biosynthesis in Mtb, identify drug targets, and develop novel, effective, safer inhibitors to treat TB in AIDS patients. Peptidoglycan (PG) is the backbone of the mycobacterial cell wall. D-alanine plays a role in the cross-linking of nascent PG strands and our central hypothesis is that D-alanine is an essential structural component of PG. Thus, for mycobacteria to survive, D-alanine must be provided in the growth medium and efficiently internalized, or made by endogenous biosynthesis. The enzyme Dalanine racemase (Alr) catalyzes the primary route of D-alanine biosynthesis in mycobacteria and the alr gene is dispensable for growth in Mycobacterium smegmatis, even in the absence of D-alanine. Unfortunately, current studies in Mtb are insufficient to determine whether Alr is an essential function of Mtb and the lethal target of the prototype inhibitor D- cycloserine (DCS), a D-alanine analog. The proposed research will allow us to determine the best strategy for drug design that either exploits Alr or targets alternative enzymes in PG biosynthesis. Thus, the conceptual innovation of our approach is to study Alr not in isolation but in the context of its niche within the Mtb metabolic network. Specifically, we will 1) Determine the essentiality of Alr in Mtb;and 2) Characterize metabolic effects of DCS treatment on Mtb. Essentiality will be analyzed both in broth cultures and human monocyte-derived macrophages. The highlight of this study will be the construction of a conditional mutant strain using a tetracycline regulated promoter to control the expression of the alr gene in Mtb. We will also use innovative Nuclear Magnetic Resonance metabolomic studies already developed in our laboratories to determine steady state metabolic pools and fluxes. The proposed studies will provide fundamental knowledge on D-alanine metabolism in Mtb. Information will be generated on the physiological essentiality and drug target potential of the Alr enzyme in this species. This project will also address the issue of whether or not there is an alternative pathway of D-alanine biosynthesis in Mtb, thus exploring the existence of a novel source of targets for further drug and/or vaccine development. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis (Mtb) is one of the most dreaded bacterial pathogens in the world, accounting for approximately 2 million deaths per year, and one of the most important causes of death in HIV-positive individuals. The world wide increase in the incidence of extensively drug-resistant strains, resistant to both first and second line drugs, underscores the urgent need to develop new anti-tuberculosis agents. D-alanine is an essential Mtb component. As an outcome of the proposed studies, we expect to determine the relevance of D-alanine racemase and related pathways of D-alanine biosynthesis to serve as targets of drug design and/or vaccine development.